Protocol

Immunoprecipitation: Denaturing Lysis

This protocol was adapted from "Immunoprecipitation," Chapter 7, in Using Antibodies by Ed Harlow and David Lane. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1999.

INTRODUCTION

For many sources of antigens, one useful method of lysis is to treat cells with harsh, denaturing solutions to release most of the protein antigens, as described here. The lysates are then diluted to reduce the denaturing conditions to levels that are suitable for the formation of antibody-antigen complexes. The resulting solution is precleared prior to immunoprecipitation. The major consideration in choosing this lysis method is that all noncovalent protein-protein interactions will be lost and that denaturation-sensitive epitopes will be destroyed. Therefore, this is not an appropriate method for many types of final assays, but it is a good method for quantitation of the antigen, careful identification of the polypeptide band recognized by the antibody, and display of denaturation-resistant epitopes.